Ionizing radiation (IR) is a known breast carcinogen in both animals and humans. IR exposure generates a variety of lesions in DNA of which the most dangerous are DNA double strand breaks (DSBs). Genomic instability can result from the presence of unrepaired DSBs leading to cell death or malignant transformation. Eukaryotes have evolved efficient systems for monitoring genomic integrity and responding rapidly to their presence via cell cycle arrest, repair, and/or apoptotic mechanisms. The master regulator of the mammalian cellular DNA DSB response pathway is the protein ATM. ATM is one of several proteins in this pathway encoded by genes implicated in breast cancer susceptibility. Despite the intriguing relationship between IR, breast cancer, and these genes, there is no clear model for how this particular biochemical pathway has specific effects on breast cancer risk. We have now developed a model for the role of ATM. In this "missense-mutation" model we propose that a subset of ATM mutations act by dominant interference, reducing the intrinsic kinase activity of ATM, and/or disruptingprotein complexes that includeATM. The model predicts that ATM-mediated risk for breast cancer is specific to carriers of this class of mutation and suggests that agents such as IR, which are potent inducers of ATM, may have enhanced carcinogenic effects in such individuals. In order to explore the complex relationship between ATM, radiation exposure and breast cancer, we initiated the WECARE (Women's Environment Cancer And Radiation Exposure) study in which 2100 women with either unilateral or asynchronous bilateral breast cancer are enrolled. In this collection, breast cancer risk factors have been assessed by questionnaire; both full radiation dosimetry reconstruction, and full mutation screening of the ATMgene have been carried out. Preliminary analysis of the ATM data reveals a significant increase in risk for second primary breast cancers in subjects who received radiation therapy and carry ATM missense mutations. However, mutation status determination was based only on consideration of sequence conservation[unreadable]no functional confirmation ofmutation status was included in the original study. In this new application, we propose to build upon these preliminary findings by directly testing the hypothesis that the incidence of contralateral breast cancer is increased among women who received radiation therapy as a treatment for their first primary breast cancer and who are carriers of specific ATM alleles which dominantly interfere with the cellular response to IR. Our studies will characterize putative ATM mutations identified in the course of WECARE screening for their functional effects on DNA damage response pathways, and then incorporate this information into the analysis of variables for the overall WECARE study.